Protein conjugates, i.e. proteins conjugated to a molecule of interest via a linker, are known in the art. For example, fluorescent labeling is a powerful technique for in vitro and in vivo visualisation, covalent immobilization of proteins is a useful strategy for industrial application and PEGylation of proteins leads to significantly enhanced circulation time. In addition, there is great interest in antibody-conjugates wherein the molecule of interest is a drug, for example a cytotoxic chemical. Antibody-drug-conjugates are known in the art, and consist of a recombinant antibody covalently bound to a cytotoxic chemical via a synthetic linker. Protein conjugates known from the prior art are commonly prepared by conjugation of a functional group to the side chain of amino acid lysine or cysteine, by acylation or alkylation, respectively.
For lysines, conjugation takes place preferentially at lysine side chains with highest steric accessibility, the lowest pKa, or a combination thereof. Disadvantage of this method is that site-control of conjugation is low.
Better control of site-specificity is obtained by alkylation of cysteines, based on the fact that typically no or few free cysteines are present in a typical protein, thereby offering the option of alkylating only those cysteines that are already present in reduced form or selectively engineered into a protein. Alternatively, cysteines can be selectively liberated by a (partial) reductive step. For example, selective cysteine liberation by reduction is typically performed by treatment of a protein with a reducing agent (e.g. TCEP or DTT), leading to conversion of a disulfide bond into two free thiols. The liberated thiols can then be alkylated with an electrophilic reagent, typically based on a maleimide chemistry, which generally proceeds fast and with high selectivity, or with haloacetamides, which also show strong preference for cysteine but side-reactions with lysine side-chains may be encountered.
A recent report (N. M. Okeley et al., Bioconj. Chem. 2013, 24, 1650, incorporated by reference herein) describes the metabolic incorporation of 6-thiofucose into the glycan of a monoclonal antibody, followed by reduction-oxidation-maleimide conjugation. Interestingly, it was found that the 6-thiofucose maleimide conjugate described above was found to display enhanced stability with respect to cysteine maleimide conjugates. However, efficiency of incorporation of 6-thiofucose was found to be only 70%.
One alternative variant of maleimide conjugation, which was applied for the generation of an antibody-drug conjugate, involves a strategy where not the nucleophilic thiol is introduced in the monoclonal antibody, but rather the maleimide. For example, T-DM1 is prepared by first (random) conjugation of lysines with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), thereby effectively charging the antibody with maleimides. In the next stage of the process, the maleimide-functionalized antibody is treated with thiol-functionalized maytansinoid, leading to the conjugate. Hence, this is a unique example where the antibody is effectively converted into an electrophilic reaction partner (instead of the common use of nucleophilic amino acid side chains for conjugation), upon treatment with SMCC. However, also in this case, by nature of the approach, only random conjugation of antibody is achieved.
Notwithstanding the versatility of the above technologies, a general disadvantage of protein conjugates obtained via alkylation with maleimides is that in general the resulting conjugates can be potentially unstable due to the reverse of alkylation, i.e. a retro-Michael reaction.
An alternative strategy to prepare conjugates of a glycoprotein, a subclass of all proteins, involves the generation of one or more aldehyde functions on the protein's glycan structure, either by chemical means (sodium periodate) or by enzymatic means (galactose oxidase). The latter aldehyde function can subsequently be employed for a selective conjugation process, for example by condensation with a functionalized hydroxylamine or hydrazine molecule, thereby generating an oxime-linked or hydrazone-linked protein conjugate, respectively. However, it is known that oximes and hydrazones, in particular derived from aliphatic aldehydes, also show limited stability over time in water or at lower pH. For example, gemtuzumab ozogamicin is an oxime-linked antibody-drug conjugate and is known to suffer from premature deconjugation in vivo.
Qasba et al. disclose in J. Biol. Chem. 2002, 277, 20833, incorporated by reference herein, that mutant galactosyltransferases GalT(Y289L), GalT(Y289I) and GalT(Y289N) can enzymatically attach GalNAc to a non-reducing GlcNAc sugar (β-benzyl-GlcNAc).
WO 2007/095506 and WO 2008/029281 (Invitrogen Corporation), incorporated by reference herein, disclose that the combination of GalT(Y289L) mutant with the C2-substituted azidoacetamido moiety 2-GalNAz-UDP leads to the incorporation of GalNAz at a terminal non-reducing GlcNAc of a glycan. Subsequent conjugation by Staudinger ligation or with copper-catalyzed click chemistry then provides the respective antibody conjugates wherein a fluorescent alkyne probe is conjugated to an antibody. WO 2007/095506 and WO 2008/029281 further disclose that trimming of the glycan can take place with endo H, thereby hydrolyzing a GlcNAc-GlcNAc glycosidic bond and liberating a GlcNAc for enzymatic introduction of GalNAz.
Qasba et al. disclose in Bioconjugate Chem. 2009, 20, 1228, incorporated by reference herein, that β-galactosidase-treated monoclonal antibodies (e.g. Rituxan, Remicade, Herceptin) having a G0 glycoform (obtained by treatment of the crude mAbs with galactosidase) are fully regalactosylated to the G2 glycoform after transfer of a galactose moiety comprising a (GalNAz) to the terminal GlcNAc residues of the glycan, leading to tetraazido-substituted antibodies, i.e. two GalNAz moieties per heavy chain. The conjugation of said tetraazido-substituted antibodies to a molecule of interest, for example by Staudinger ligation or cycloaddition with an alkyne, is not disclosed. The transfer of a galactose moiety comprising a C2-substituted keto group (C2-keto-Gal) to the terminal GlcNAc residues of a G0 glycoform glycan, as well as the linking of C2-keto-Gal to aminooxy biotin, is also disclosed. However, as mentioned above, the resulting oxime conjugates may display limited stability due to aqueous hydrolysis.
A disadvantage of the methods disclosed in WO 2007/095506, WO 2004/063344 and Bioconjugate Chem. 2009, 20, 1228 is that the conjugates obtained by azide-alkyne click chemistry in all cases feature a triazole linkage, which may be disadvantageous with respect to immunogenicity of the ADC. Moreover, in case copper-catalyzed click chemistry is employed, protein damage resulting from undesired oxidative processes may occur, as is disclosed in e.g. Hong et al., Angew. Chem. Int. Ed. Engl. 2009, 48, 9879 (incorporated by reference).
Based on the above, it is clear that galactose can be introduced to proteins featuring a terminal GlcNAc-moiety upon treatment with wild type Gal-T1/UDP-Gal (leading to Gal-GlcNAc-protein), while N-acetylgalactosamine can be introduced upon treatment with GalT1 mutant Y289L (affording GalNAc-GlcNAc-protein). It has also been shown by Elling et al. (ChemBioChem 2001, 2, 884, incorporated by reference herein) that a variety of human galactosyltransferases (β4-Gal-T1, β4-Gal-T4 and β3-Gal-T5), but not bovine 134-Gal-T1, can accommodate a 6-biotinylated modification of galactose in UDP-Gal, in the absence of Mn2+, leading to effective transfer to model proteins BSA-(GlcNAc)17 and ovalbumin. Similarly, Pannecoucke et al. (Tetrahedron Lett. 2008, 49, 2294, incorporated by reference herein) demonstrated that commercially available bovine β4-Gal-T1 under standard conditions is also able to transfer UDP-6-azidogalactose to a model GlcNAc-substrate, but the transfer to a GlcNAc-protein was not demonstrated.
Based on the above, it may be concluded that a strategy involving chemoselective or enzymatic modification of glycans on glycoproteins is a versatile strategy for the site-specific preparation of protein conjugates. However, the current technologies generate linkages of unpredictable stability (maleimides, oximes, hydrazones) or unnatural constitution (triazoles). Moreover, the mode of preparation may be slow and require large excesses of reagent (oxime or hydrazone ligation) or may lead to protein damage (copper-catalyzed click chemistry). Finally, the recently disclosed strategy for metabolic thiofucose incorporation has promise, but efficiency of thiofucose incorporation is low (±70%) due to competition with natural fucose.
Conjugations of biomolecules based on thiols are well-known in the art. In particular, reaction of thiols with maleimide is a fast and selective process, which typically rapidly leads to the desired conjugate. Less popular but also regularly applied are halogenated acetamides that may also react with high selectivity with free thiols although chemoselectivity is compromised with respect to maleimide conjugation. A particular advantage of conjugation with halogenated acetamides is the irreversible formation of a thioether, which compares favorably to maleimide conjugates with respect to stability. The latter stability also applied to conjugates formed by reaction of thiols with allenamide, as most recently reported by Abbas et al., Angew. Chem. Int. Ed. 2014, 53, 7491-7494, incorporated by reference. Other alternatives for conjugation to thiols are also known, for example vinylsulfone conjugation, but less frequently applied. Finally, light-induced thiol-ene reaction has also been shown to be suitable for protein conjugation, see for example Kunz et al. Angew. Chem. Int. Ed. 2007, 46, 5226-5230), also in this case leading to highly stable thioethers.